Highly thermostable fluorescent proteins

ABSTRACT

Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99° C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80° C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

RELATED APPLICATIONS

This patent application claims the benefit of the filing date of UnitedStates Provisional Patent Application No. 61/008,689 filed Dec. 21,2007.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.DE-AC52-06 NA 25396 awarded by the U.S. Department of Energy. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

GFP and its numerous related fluorescent proteins are now in widespreaduse as protein tagging agents (for review, see Verkhusha et al., 2003,GFP-like fluorescent proteins and chromoproteins of the class Anthozoa.In: Protein Structures: Kaleidescope of Structural Properties andFunctions, Ch. 18, pp. 405-439, Research Signpost, Kerala, India).GFP-like proteins are an expanding family of homologous, 25-30 kDapolypeptides sharing a conserved 11 beta-strand “barrel” structure. TheGFP-like protein family currently comprises well over 100 members,cloned from various Anthozoa and Hydrozoa species, and includes red,yellow and green fluorescent proteins and a variety of non-fluorescentchromoproteins. A wide variety of fluorescent protein labeling assaysand kits are commercially available, encompassing a broad spectrum ofGFP spectral variants and GFP-like fluorescent proteins, including DsRedand other red fluorescent proteins (Clontech, Palo Alto, Calif.;Amersham, Piscataway, N.J.).

However, the stability of fluorescent proteins is limited. Variousapproaches aimed at stabilizing fluorescent proteins have beenundertaken. For example, Siemering et al. described the generation of aGFP mutant (GFPA) using site-directed mutagenesis, reporting that themutant showed reduced sensitivity to temperature in both bacteria andyeast cultured at 37° C. (Siemering et al., 1996, Curr Biol 6: 1653).U.S. Pat. No. 6,414,119 described a GFP mutant showing modestimprovements in thermal stability over wild type GFP (reportedlyretaining fluorescence and solubility at 42° C., and showing somefluorescence at 50° C.). More recently, Pedelacq et al.,⁷ used directedevolution to increase the stability of GFP by selecting for resistanceto the destabilizing effects of a poorly folding and aggregatingferritin sequence fused upstream. The first fusions were very weaklyfluorescent, but with further evolution of the GFP, this externaldestabilization could be overcome and a variant (termed “superfolderGFP”) able to resist the folding interference of ferritin was selected.This was shown to be considerably more stable than standard GFP by anumber of different measures, including resistance to thermal andchemical denaturation.

A number of different methods have been developed to create thermostableproteins, most of which involve the creation of libraries and theidentification of improved proteins by selection or screening.Conceptually, the most straightforward way to identify proteins withimproved thermostability has been to apply a thermal challenge to acollection of individual clones and test the remaining functionality ofthe clones, repeating this process if necessary, to combine usefulmutations⁸⁻¹⁰. A similar method, which does not rely on such extensivescreening requirements, involves direct selection of clones growing atelevated temperature within thermophilic bacteria. However, to date,this method has only been applied to the selection of thermophilicantibiotic resistance proteins^(11, 12), and as laboratory organismstypically do not grow at elevated temperatures, it has been difficult togeneralize. As a result, considerable effort has been put into thedevelopment of alternative approaches which involve selection orscreening for biophysical or biological properties which can serve assurrogates for, and are often correlated with, thermostability.

One of the first examples of this approach is the PROSIDE (proteinstability increased by directed evolution)¹³⁻²⁰ approach in whichresistance to protease digestion is used as the surrogate property forprotein stability, with filamentous phage infectivity being theselection modality. Proteins under test are expressed between twodomains in g3p (the phage receptor for bacteria): if they are cleaved byprotease, the filamentous phage loses the N terminal g3p domain andconsequently its ability to infect: if the protein is protease resistantinfectivity is maintained. This has been successfully used to increasethe stability of the betel domain of protein G¹⁵, the cold shock proteinof B. subtilis ¹⁷ and ribonuclease T1¹³. In another approach involvingdirected evolution, Shusta et al., showed that the display levels ofheterologous proteins on the surface of yeast correlated with expressionlevels and thermal stability²¹, although exceptions to this have beenrecently described²².

Consensus engineering^(23, 24) is an approach to increase proteinstability which does not use directed evolution, but the informationalcontent of aligned sequences. By modifying a sequence so that it moreclosely resembles a consensus derived from the alignment of numerousproteins of a particular family, it has been found that significantincreases in stability can be obtained. This has been applied toantibodies and antibody fragments^(5, 24-31), GroELminichaperones^(32, 33), p53³⁴, WW³⁵ and SH3 domains³⁶. More recentlyconsensus engineering, has been applied to the creation of novelproteins, rather than the stepwise modification of pre-existing ones toresemble a consensus. Perhaps the most striking success was theapplication to phytases³⁷⁻⁴⁰, in which a final protein with a Tm of90.4° C. was obtained: 52° C. greater than the best component parentalsequence⁴⁰. Similar stability was obtained with a consensus ankyrinsequence based on the alignment of 2000 different ankyrins⁴¹⁻⁴³. Werecently applied this method to the creation of a consensus greenprotein (CGP)⁴⁴.

Although we obtained a functional fluorescent protein, its Tm was 5° C.less than the monomeric Azami Green⁴⁵ used to identify the sequencescomprising the consensus. However, in this case no effort was made toexamine the effects of individual mutations, and it is likely that someof the consensus mutations were destabilizing, as had been previouslyshown for the phytase³⁷⁻⁴⁰.

Other methods used to increase protein stability, relying heavily onstructural information, include “helix capping”⁴⁵⁻⁴⁹ oroptimization⁵⁰⁻⁵², the introduction of salt bridges or their replacementby hydrophobic interactions⁵³⁻⁵⁹, the introduction of clusters ofaromatic-aromatic interactions⁶⁰⁻⁶² and rigidification strategies, inwhich disulfide bonds or glycine to alanine, or Xaa to proline changesare introduced⁶³⁻⁶⁵. However, most of these have been carried out onmodel structures, and none has been widely adopted.

Thermostabilization of proteins is regarded as important in a number ofbiotechnological and pharmaceutical applications. Within the context ofindustrial enzymes, thermostability leads to longer enzyme survivaltimes, as well as more efficient reactions at higher temperatures anddiminished microbial contamination, all of which result in diminishedcosts, while in the pharmaceutical arena, thermostability of proteintherapeutics leads to longer half lives and more effective drugs¹⁻³.Thermostability has also been regarded as important in the use ofproteins as scaffolds to generate libraries of specific binders. It hasbeen reasoned that if a starting scaffold is more stable, it will bemore tolerant to the destabilizing effects of mutations, or insertions,used to mediate binding. This has been shown for affinity reagents basedon ankyrins⁴, and has also been applied to the creation of phageantibody libraries⁵. Finally, proteins of increased thermostability aremore resistant to mutations than the protein from which they arederived, promoting evolvability by providing greater permissivity tomutations leading to novel functions⁶⁻⁷.

SUMMARY OF THE INVENTION

The invention relates novel and highly thermostable fluorescent proteins(TSFPs), methods for generating these and other stability-enhancedproteins, polynucleotides encoding such proteins, and assays and methodfor using the TSFPs and TSFP-encoding nucleic acid molecules of theinvention. Exemplary TSFPs are provided. In particular, polypeptidescomprising eCGPs of the invention, including but not limited to thosehaving the sequences of SEQ ID NOS: 9 and 10, are provided.Additionally, nucleic acid molecules comprising a polynucleotideencoding such polypeptides are also provided, and include withoutlimitation, nucleic acid molecules which comprise polynucleotidesencoding the sequences of SEQ ID NOS: 4 and 5. Vectors comprising suchnucleic acid molecules are also provided, as are cells comprising suchvectors.

The invention also provides a method for generating stability-enhancedvariants of a protein, including but not limited to fluorescentproteins. The method of the invention is described, infra, and in theExamples which follow. Briefly, in a simplified description, the methodentails internally destabilizing the protein using a heterologousinsertion, evolving the protein sequences adjacent to the heterologousinsertion to overcome the destabilization, and then removing theheterologous insert.

The TSFPs of the invention show extremely enhanced levels of stabilityand thermotolerance. In one case, for example, a TSFP of the inventionis so stable it can be heated to 99° C. for short periods of timewithout denaturing, and retains 85% of its fluorescence when heated to80° C. for several minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic outline of the internal destabilization methodologyused to generate thermostable variants of CGP, an artificial fluorescentprotein (Dai et al., 2007, supra). (A) strategy applied to CGP. Afterthe insertion of a single insert, CGP loses fluorescence which wasregained by mutation and selection. This process was repeated untilfluorescent proteins resistant to the destabilizing effects of threeloops were obtained. For each evolved fluorescent variant, a gene issynthesized which lacks the inserted loop(s). (B) shows inducedbacterial colonies, with a colony expressing CGP before (colony 1) andafter insertion of the HCDR3 in loop 1 (colony 2) or loop 2 (colony 3).The remaining colonies are mutated variants, showing the improvement influorescence.

FIG. 2. Accumulation of amino acid mutations through iterative internaldestabilization. The original sequence of CGP is represented by thesmall squares at the top, with the positions of amino acids thatunderwent mutation indicated as black boxes. The exact positions andwild type sequences of these are shown below, with the three insertionsites indicated as inverted red triangles flanked by the exactpositions. The mutations occurring at each site, for each evolutionaryround and loop insertion strategy, are shown. White squares indicatewild type sequence. Where a mutation has occurred, the letter indicatesthe new mutation, and the number the percentage of the sequencedfluorescent clones that contain that mutation. This is also representedgraphically by that portion of the white square colored green. Forexample, at position 7, in all early evolutionary rounds 100% of cloneschanged the wild type aspartate to a glutamate. If more than onemutation is found at a particular site, both amino acids are given withtheir percentages, indicated by green and yellow boxes. After threerounds when loops 2 and 3 were targeted, 12% of clones also showed avaline at this position, which increased to 50% in later rounds. Thepercentage of clones carrying a particular mutation are shown if thatmutation comprises more than 5% of clones in any of the evolutionaryrounds.

FIG. 3. Sequence alignments of various. TSFPs of the invention, comparedto the reference protein, CGP, and to the protein from which CGP wasinitially derived, mAG (BAD52002). Shown are sequences of CGP [SEQ IDNO: 27], eCGP1 [SEQ ID NO: 6], eCGP13 [SEQ ID NO: 8], eCGP2 [SEQ ID NO:7], eCGP23 [SEQ ID NO: 9], eCGP123 [SEQ ID NO: 10].

FIG. 4. Absorption and emission of purified TSFPs. (A) showspurification and expression levels of the different purified proteins.The amounts given correspond to the total amount of purified proteinfrom 60 ml fermentation volume. (B) shows absorption and emission of thepurified CGP, various eCGP proteins, and mAG normalized to 1 for therespective peaks. Peak values are provided in TABLE I.

FIG. 5. Thermal stability of evolved fluorescent proteins. (A)Fluorescence profile of the different proteins gradually heated to 99°C. and then allowed to recover at 30° C. Fluorescence was measured everysix seconds, and normalized to the fluorescence level at 30° C. (B)Enlargement of fluorescence profile from 90-99° C., showing thepersistence of low levels of fluorescence with eCGP123 and eCGP23 at 99°C. (C) Stability with repeated heating and cooling cycles. Proteins wereheated to 99° C. for one minute and then cooled to 30° C. for twominutes. This was carried out sixty times and fluorescence was measuredat the end of each heating or cooling period. (D) The survival offluorescent proteins at 80° C. was assessed by heating to 80° C.,measuring fluorescence every six seconds. Fluorescence was normalized tothe fluorescence level after five minutes at 80° C., at which time theinitial rapid loss of fluorescence due to heating stabilized. (E) Thesurvival of fluorescent proteins at 80° C. was assessed by heating to80° C., and measuring fluorescence each six seconds. Fluorescence wasnormalized to the fluorescence level after five minutes at 80° C. (FIG.5D). (F): As FIG. 5E, except proteins were heated to 85° C.

FIG. 6. Stability to chemical denaturation. (A): Each of the fluorescentproteins was diluted into 48 different, Guanidium hydrochlorideconcentrations, with 7.4 M being the highest concentration. The residualfluorescence was measured at equilibrium, normalized and plotted. Therecovered fluorescence was normalized by dividing the fluorescence ofcorresponding non-denatured samples diluted in parallel. (B): Dependenceof the standard free energy of denaturation on guanidine concentrationassuming a two-state folding model for the fluorescent proteins (TABLEIII). (C): Refolding kinetics. Long-term (2000 s) progress curves forrecovery of fluorescence during refolding of Gdn HCl-denatured eCGP123(blue), CGP (magenta), and mAG (green) upon 20-fold dilution ofdenatured samples in fresh buffer containing 1 mM DTT at 25° C. (seeMethods), with the inset showing the short-term progress curves. Initialrates V_(I) were obtained from slope at t=0 s of 2^(nd)-orderpolynomials fitted to the first 12 s of short-term progress curves.Fluorescence normalized by dividing by final fluorescence value at 15 h.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and CurrentProtocols in Molecular Biology (Ausbel et al., eds. John Wiley & Sons.Inc. 2001. As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof (“polynucleotides”) in eithersingle- or double-stranded form. Unless specifically limited, the term.“polynucleotide” encompasses nucleic acids containing known analogues ofnatural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g. degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19: 5081;Ohtsuka et al., 1985 J. Biol. Chem. 260: 2605-2608; and Cassol et al.,1992; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98). The termnucleic acid is used interchangeably with gene, cDNA, and mRNA encodedby a gene.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts at al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 25 to approximately 500 amino acids long. Typical domains aremade up of sections of lesser organization such as stretches of β-sheetand α-helices. “Tertiary structure” refers to the complete threedimensional structure of a polypeptide monomer. “Quaternary structure”refers to the three dimensional structure formed by the noncovalentassociation of independent tertiary units. Anisotropic terms are alsoknown as energy terms.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Forexample, one type of vector is a plasmid, a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a phage vector. Another type of vector is a viral vector,wherein additional DNA segments may be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated into,the genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” or “expression vectors”.

The term “host cell” (or “recombinant host cell”), as used herein,refers to a cell that has been genetically altered, or is capable ofbeing genetically altered by introduction of an exogenouspolynucleotide, such as a recombinant plasmid or vector, and includesnot only the particular subject cell but also the progeny thereof.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term “host cell” as used herein.

The term “link” as used herein refers to a physical linkage as well aslinkage that occurs by virtue of co-existence within a biologicalparticle, e.g. phage, bacteria, yeast or other eukaryotic cell.

“Physical linkage” refers to any method known in the art forfunctionally connecting two molecules (which are termed “physicallylinked”), including without limitation, recombinant fusion with orwithout intervening domains, intein-mediated fusion, non-covalentassociation, covalent bonding (e.g., disulfide bonding and othercovalent bonding), hydrogen bonding; electrostatic bonding; andconformational bonding, e.g., antibody-antigen, and biotin-avidinassociations.

“Fused” refers to linkage by covalent bonding.

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as VH and VL genes orpolypeptides (i.e., in a scFv), and serves to place the two molecules ina preferred configuration.

The term “isolated” refers to material which is substantially oressentially free from components which normally accompany the materialas it is found in its native or natural state. However, the term“isolated” is not intended refer to the components present in anelectrophoretic gel or other separation medium. An isolated component isfree from such separation media and in a form ready for use in anotherapplication or already in use in the new application/milieu. An“isolated” antibody is one that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The terms “label” and “detectable label” refer to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” or “detectably labeled” antibody.The label may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable. A great number of such labels are known in the art,including without limitation protein tags, radioisotopes, metalchelators, enzymes, fluorescent compounds (dyes, proteins, chemicals),bioluminescent compounds, and chemiluminescent compounds.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, a nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a nucleic acid encoding afluorescent protein from one source and a nucleic acid encoding apeptide sequence from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95%identity over a specified region, when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the compliment of a testsequence. Preferably, the identity exists over a region that is at leastabout 22 amino acids or nucleotides in length, or more preferably over aregion that is 30, 40, or 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homologyalignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443,by the search for similarity method of Pearson & Lipman, 1988, Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP. BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al, 1977, Nuc.Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 are used, typically withthe default parameters described herein, to determine percent sequenceidentity for the nucleic acids and proteins of the invention. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10. M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, 1993,Proc. Nat'l. Acad. Sci. USA. 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “as determined by maximal correspondence” in the context ofreferring to a reference SEC) ID NO means that a sequence is maximallyaligned with the reference SEQ ID NO over the length of the referencesequence using an algorithm such as BLAST set to the default parameters.Such a determination is easily made by one of skill in the art.

The invention provides novel and highly stable fluorescent proteins.Because the proteins of the invention are particularly stable at veryhigh temperatures, they have been termed Thermostable FluorescentProteins, or “TSFPs”. Several green TSFPs are disclosed herein, as wellas a unique methodology for generating such variants of other proteins,including fluorescent proteins. In particular embodiments disclosedherein, a class of TSFPs termed eCGPs are provided. The eCGPs of theinvention were derived from an artificial fluorescent protein previouslygenerated using a consensus engineering approach (consensus greenfluorescent protein, or CGP⁴⁴). The invention also providespolynucleotides encoding eCGPs, as well as vectors comprising suchpolynucleotides and cells transformed or containing such vectors.Various assay methods which utilize the TSFPs of the invention are alsoencompassed by the invention.

The TSFPs of the invention may be employed for all applications, methodsand uses to which GFP and other fluorescent proteins are or may beapplied, including their use as markers, as protein tags, in solubilityscreening, in the generation of split-CGP systems and assays, in proteintrafficking and localization assays, in applications involving FRET, andthe like. For example, TSFPs may be coupled to antibodies,polynucleotides or other receptors for use in detection assays such asimmunoassays or hybridization assays, or to track the movement ofproteins in cells. TSFPs may also be useful in systems to detectinduction of transcription. For example, a nucleotide sequence encodinga TSFP can be fused to a promoter or other expression control sequenceof interest, which can be contained in an expression vector, theconstruct can be transfected into a cell, and induction of the promoter(or other regulatory element) can be measured by detecting the presenceor amount of fluorescence, thereby allowing a means to observe theresponsiveness of a signaling pathway from receptor to promoter.

Because the TSFPs of the invention demonstrate remarkable stability in anumber of challenging environments, they may find use in processes,assays and other applications in which a high degree of stability isrequired in order for the fluorescent phenotype to survive. For example,eCGPs show a remarkable degree of thermotolerance. Some of the eCGPs,such as eCGP23 and eCGP123, are able to retain fluorescence after beingexposed to very high temperatures. For example, both eCGP23 and eCGP123are able to recover almost completely after heating at 99° C., atemperature that irreversibly destroys folding in all other fluorescentproteins tested. Similarly, both of these eCGPs are able to retain somedegree of fluorescence even at the high temperature of 99° C.Additionally, both of these eCGPs retain approximately 85% of theirambient temperature fluorescence levels for at least 14 hours at 80° C.Thus, these two eCGPs demonstrate remarkable and hitherto unreportedlevels of thermotolerance for fluorescent proteins, and should find usein various applications for which thermostability as well as increasedstability generally are required. Detailed characterization of eCGPs isprovided in the Examples which follow.

The fluorescence loss that occurs when fluorescent proteins are heatedis caused by a combination of disruption of the local fluorophoreenvironment caused by thermal vibrations and unfolding. Unfolding, inturn, can be either reversible or irreversible. In general, the loss offluorescence caused by thermal vibrations is almost instantaneouslyreversible, that caused by reversible unfolding depends upon thekinetics of refolding, while irreversible unfolding does not recover. Anumber of lines of evidence indicate that most of the loss offluorescence with heating eCGP123 to 99° C. is due to disruption of thelocal fluorophore environment, rather than unfolding of the protein.First, a thermal melt does not show the inflection point characteristicof the onset of cooperative unfolding, shown by the other proteins(FIGS. 5 a and 5 b); secondly, at 99° C., some residual fluorescence isclearly present for eCGP123 and eCGP23, while it is completely lost forthe other proteins (FIG. 5 b and 5 c); thirdly, upon cooling after thethermal melt, over 60% of the fluorescence returns immediately (FIG. 5 aand TABLE II); and finally, when the protein is repeatedly cycledbetween 99 and 30° C. fluorescence recovery is essentially immediate,and complete, with each cycle (FIG. 5 c), while refolding would beexpected to take longer. However, although it appears that most of theprotein remains folded after short periods at 99° C., it is clear thatprolonged incubations at high temperatures below 99° C. can causesignificant loss of fluorescence. After 14 hours at 80° C., only 15% ofthe fluorescence normalized after stabilization at 80° C. is lost (FIG.5 e), whereas at 85° C., only 15% of the fluorescence remains (FIG. 5f).

When the thermal stability of the different evolved eCGP proteins iscompared, the increased stability with increased evolution is striking,with the order of stability being eCGP123>eCGP23>eCGP13>eCGP2>eCGP1>CGP:evolution around each additional loop, results in, increased stability.However, the individual loops are not equal in their stabilizingeffects, with evolution around loop 2, appearing to provide the greatestindividual stabilizing effect (compare eCGP1 to eCGP2 and eCGP13 toeCGP23). In fact, eCGP23 and eCGP123 are extremely similar to oneanother in their stability.

The eCGPs were also characterized by chemical denaturation usingdifferent concentrations of guanidine hydrochloride (FIG. 6 a). Seealso, Example 1, infra.

The invention further provides a method for generatingstability-enhanced proteins. The method by which the eCGPs of theinvention were generated is explained in detail in the Examples whichfollow. This method may be applied to other fluorescent proteins, andindeed, to virtually any protein, in order to generateincreased-stability variants. Briefly, in the method of the invention, arecursive directed evolution strategy is employed, in which singledestabilizing inserts are grafted into exposed loops of the protein insuch a way that upon each insertion, folding and function aresignificantly affected but not destroyed (FIG. 1A). Upon overcoming theeffect of a single insert by the initial round of evolution, theprocedure is repeated with additional destabilizing inserts in aniterative fashion. The method enables one to overcome a finaldestabilizing force that would completely destroy both folding andfunction if applied in a single step.

The application of this method to a fluorescent protein is facilitatedby the ease with which screening for correct folding can be carried out.However, this method is likely to be generally applicable to anyprotein, providing three criteria are fulfilled: 1) Surface exposedinsert sites are correctly identified; 2) An appropriate destabilizinginsert is used; and 3) A method to select correctly folded clones isavailable. In the example used herein, the structure of mAG, which wasused to derive CGP, allowed the modeling and identification of thesurface exposed loops. Although this is the ideal situation, when astructure or model are not available, it is possible that theapplication of secondary^(75, 76) or tertiary structural predictionmethods^(77, 78) may provide sufficient information to identify suitablesurface turns, since it is extremely unlikely that inserts placed withinthe protein core could be overcome by any degree of evolution.

The destabilizing insert used to generate the exemplified TSFPsdisclosed herein here was based on an antibody heavy chaincomplementarity determining region 3 (HCDR3). This insert was chosen asthe N and C termini of HCDR3s are close to one another within thecontext of an anti-parallel beta strand⁶⁷, thereby presumptivelyproviding destabilization without completely inhibiting folding. It islikely that alternative inserts could also provide appropriate degreesof destabilization, and it is possible that a panel of destabilizinginserts could be developed. In fact, such inserts could even comprisewhole proteins in which the N and C termini were close to one another.

In the practice of the method of the invention, it is important toidentify or develop a method to select or screen for correctly foldedclones. When applied to fluorescent proteins, it is relativelystraightforward to examine bacterial clones for fluorescence. A similarapproach could be used for enzymes which can be expressed in bacteria,and for which calorimetric or fluorescent reagents are available.However, for the majority of proteins for which there is no obviousdirectly screenable phenotype, a separate screen for correct folding isrequired. This is not unlike the use of phage⁷⁹⁻⁸¹ or yeast display⁸²⁻⁸⁵to identify amino acids comprising specific binding sites: it is notsufficient to identify clones no longer binding to the binding partner,since loss of binding may be due to lack of folding. In addition to thenegative selection for loss of binding, a positive selection for correctfolding must also be included in the selection strategy. In the case ofyeast, it is relatively straightforward, as only correctly foldedproteins reach the cell surface and poorly folding proteins are retainedin the endoplasmic reticulum. As a result it is sufficient to detectsurface display using monoclonal⁸³, polyclonal⁸² or anti-tag⁸⁴antibodies. In the case of phage display, recognition of conformationalepitopes.

The invention also provides various methods which utilize TSFPs and TSFPcoding sequences, such methods being currently employed with variousother fluorescent proteins and variants thereof. For example, theinvention provides a method for identifying the presence of a moleculein a sample. Such a method can be performed, for example, by linking afluorescent protein variant of the invention to the molecule, anddetecting fluorescence due to the fluorescent protein variant in asample suspected of containing the molecule. The molecule to be detectedcan be a polypeptide, a polynucleotide, or any other molecule,including, for example, an antibody, an enzyme, or a receptor, and thelike. The sample to be examined can be any sample, including abiological sample, an environmental sample, or any other sample forwhich it is desired to determine whether a particular molecule ispresent therein.

TSFPs may be linked to the molecule directly or indirectly, using anylinkage that is stable under the conditions to which theprotein-molecule complex is to be exposed. Thus, a TSFP and the subjectmolecule can be linked via a chemical reaction between reactive groupspresent on the protein and molecule, or the linkage can be mediated bylinker moiety, which contains reactive groups specific for thefluorescent protein and the molecule. It will be appreciated that theappropriate conditions for linking a TSFP and the molecule are selecteddepending, for example, on the chemical nature of the molecule and thetype of linkage desired. Where the molecule of interest is apolypeptide, a convenient means for linking a TSFP and the molecule isby expressing them as a fusion protein from a recombinant nucleic acidmolecule, which comprises a polynucleotide encoding, for example, aneCGP operatively linked to a polynucleotide encoding the polypeptidemolecule.

TSFPs may also be used in methods to identify, agents and/or conditionsthat regulate the activity of an expression control sequence. Suchmethods may be performed, for example, by exposing a recombinant nucleicacid molecule, which includes a polynucleotide encoding a TSFPoperatively linked to an expression control sequence, to an agent orcondition suspected of being able to regulate expression of apolynucleotide from the expression control sequence, and detectingfluorescence of the TSFP due to such exposure. Such methods may beuseful for identifying chemical or biological agents, including cellularproteins, that can regulate expression from the expression controlsequence, including cellular factors involved in the tissue specific,expression from the regulatory element. As such, the expression controlsequence can be a transcription regulatory element such as a promoter,enhancer, silencer, intron splicing recognition site, polyadenylationsite, or the like; or a translation regulatory element such as aribosome binding site.

The invention also provides conservatively modified variants, as will beunderstood the those skilled in the art. Conservative substitutions maybe tested using assays described herein or otherwise well known in theart. Other eCGP variant proteins can be identified, for example, usingmethods described in WO0123602 and other methods to select for increasedfolding. For example, to obtain an eCGP variant with increased foldingability, a “bait” or “guest” peptide that decreases the folding yield ofthe eCGP is linked to the eCGP. The guest peptide can be any peptidethat, when inserted, decreases the folding yield of the eCGP, which maybe measured by fluorescence, for example. A library of mutatedfluorescent proteins is created. The bait peptide is inserted into theeCGP and the degree of fluorescence of the protein is assayed. Thoseclones exhibit increased fluorescence relative to a fusion proteincomprising the bait peptide and parent eCGP are selected (thefluorescent intensity reflects the amount of properly folded fluorescentprotein). The guest peptide may be linked to the eCGP at an end, or maybe inserted at an internal site.

Various techniques for introducing mutations are well known in the art.These include, but are not limited to, such techniques as error-pronePCR, chemical mutagenesis, and cassette mutagenesis. Alternatively,mutator strains of host cells may be employed to add mutationalfrequency (Greener and Callahan, 1995, Strategies in Mol. Biol. 7: 32).For example, error-prone PCR (see, e.g., Ausubel, supra) useslow-fidelity polymerization conditions to introduce a low level of pointmutations randomly over a long sequence. Other mutagenesis methodsinclude, without limitation, recombination, oligonucleotide-directedmutagenesis, phosphothioate-modified DNA mutagenesis, mutagenesis usinguracil-containing templates, mutagenesis using gapped duplex DNA, pointmismatch repair, mutagenesis using repair-deficient host strains, anddeletion mutagenesis. Kits for mutagenesis are commercially available(e.g., Bio-Rad, Amersham International). More recent approaches includecodon-based mutagenesis, in which entire codons are replaced, therebyincreasing the diversity of mutants generated, as exemplified by the RIDmethod described in Murakami et al., 2002, Nature Biotechnology, 20:76-81.

The TSFP polypeptides may be prepared using methods well known in theart, including by peptide synthesis and recombinant production means.For example, an eCGP may be synthesized according to standardsolid-phase methodologies, utilizing the amino acid sequences providedherein, such as may be performed on an Applied Biosystems Model 430Apeptide synthesizer (Applied Biosystems, Foster City, Calif.), accordingto manufacturer's instructions. Other methods of synthesizing peptidesor peptidomimetics, either by solid phase methodologies or in liquidphase, are well known to those skilled in the art.

Also provided are vectors containing the TSFP polynucleotides of theinvention, as well as host cells transformed or transfected with, orotherwise made to contain, such vectors. Also provided is a recombinantnucleic acid molecule, which includes at least one polynucleotideencoding a TSFP operatively linked to one or more other polynucleotides.The one or more other polynucleotides can be, for example, atranscription regulatory element such as a promoter or polyadenylationsignal sequence, or a translation regulatory element such as a ribosomebinding site. Such a recombinant nucleic acid molecule can be containedin a vector, which can be an expression vector, and the nucleic acidmolecule or the vector can be contained in a host cell. A vector of theinvention will generally contain various elements required forreplication in a prokaryotic or eukaryotic host system, or both, asrequired. Such vectors, which include plasmid vectors and viral vectorssuch as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus,vaccinia virus, semliki forest virus and adeno-associated virus vectors,are well known and can be purchased from a number of commercial sourcesor constructed using methods well known in the art.

The disclosed eCGPs, eCGP variants, or fusions of an eCGP and anotherpolypeptide, may conveniently expressed in a suitable host cell, such asan E. coli cell, using an eCGP-encoding polynucleotide, such as the DNAcoding sequences for eCGPs provided in the TABLE OF SEQUENCES, infra.

There are many expression systems for producing the proteins of theinvention that are well know to those of ordinary skill in the art.(See, e.g., Gene Expression Systems, Fernandes and Hoeffler, Eds.Academic Press, 1999; Russell & Sambrook, supra). Commonly usedprokaryotic control sequences, which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, include such commonly usedpromoters as the beta-lactamase (penicillinase) and lactose (lac)promoter systems, the tryptophan (trp) promoter, the tac promoter andthe lambda-derived P_(L) promoter and N-gene ribosome binding site. Theparticular promoter system is not critical to the invention, anyavailable promoter that functions in prokaryotes can be used. Standardbacterial expression vectors include plasmids such as pET, pTET,pBR322-based plasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, λ-phage derivedvectors, p15A-based vectors and fusion expression systems such as GST.Epitope tags can also be added to recombinant proteins to provideconvenient methods of isolation, e.g., c-myc, HA-tag, 6-His tag, maltosebinding protein, VSV-G tag, anti-DYKDDDDK tag, or any such tag, a largenumber of which are well known to those of skill in the art.

For expression of fusion polypeptides in prokaryotic cells other than E.coli, regulatory sequences for transcription and translation thatfunction in the particular prokaryotic species is required. Suchpromoters can be obtained from genes that have been cloned from thespecies, or heterologous promoters can be used. For example, the hybridtrp-lac promoter functions in Bacillus in addition to E. coli. These andother suitable bacterial promoters are well known in the art and aredescribed, e.g., in Russell & Sambrook and Ausubel et al. Bacterialexpression systems for expressing the proteins of the invention are wellknown and commercially available.

Similarly, the for expression of fusion polypeptides in eukaryoticcells, transcription and translation sequences that function in theparticular eukaryotic species are required. For example, eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available. In yeast, vectorsinclude Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicatingplasmids (the YRp series plasmids) and pGPD-2. Expression vectorscontaining regulatory elements from eukaryotic viruses are typicallyused in eukaryotic expression vectors, e.g., SV40 vectors, papillomavirus vectors, and vectors derived from Epstein-Barr virus. Otherexemplary eukaryotic vectors include those employing the CMV promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Either constitutive or regulated promoters can be used in the presentinvention. Regulated promoters can be advantageous because the hostcells can be grown to high densities before expression of thepolypeptide is induced. High level expression of heterologous proteinsslows cell growth in some situations. An inducible promoter is apromoter that directs expression of a gene where the level of expressionis alterable by environmental or developmental factors such as, forexample, temperature, pH, anaerobic or aerobic conditions, light,transcription factors and chemicals.

Inducible promoters for other organisms are also well known to those ofskill in the art. These include, for example, the metallothioneinpromoter, the heat shock promoter, as well as many others.

Translational coupling may be used to enhance expression as is wellknown. The strategy uses a short upstream open reading frame derivedfrom a highly expressed gene native to the translational system, whichis placed downstream of the promoter, and a ribosome binding sitefollowed, after a few amino acid codons by a termination codon. Justprior to the termination codon is a second ribosome binding site, andfollowing the termination codon is a start codon for the initiation oftranslation. The system dissolves secondary structure in the RNA,allowing for the efficient initiation of translation.

The construction of polynucleotide constructs generally requires the useof vectors able to replicate in host bacterial cells, or able tointegrate into the genome of host bacterial cells. Such vectors arecommonly used in the art. A great number of systems and kits arecommercially available for the purification of plasmids from bacteria(for example, EasyPrepJ, FlexiPrepJ, from Pharmacia Biotech;StrataCleanJ, from Stratagene; and, QIAexpress Expression System,Qiagen). The isolated and purified plasmids can then be furthermanipulated to produce other plasmids, and used to transform cells.

The TSFP polypeptides can be expressed intracellularly, or can besecreted from the cell. Intracellular expression often results in highyields. If necessary, the amount of soluble, active fusion polypeptidemay be increased by performing refolding procedures (see, e.g. Sambrooket al., supra.: Marston at al., Bio/Technology (1984) 2:800; Schoner atal., Bio/Technology (1985) 3: 151). Fusion polypeptides of the inventioncan be expressed in a variety of host cells, including E. coli, otherbacterial hosts, yeast; and various higher eukaryotic cells such as theCOS, CHO and HeLa cells lines and myeloma cell lines. The host cells canbe mammalian cells, insect cells, or microorganisms, such as, forexample, yeast cells, bacterial cells, or fungal cells.

Once expressed, the recombinant polypeptides can be purified accordingto standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)).

To facilitate purification of the TSFP polypeptides of the invention,the nucleic acids that encode the fusion polypeptides can also include acoding sequence for an epitope or “tag” for which an affinity bindingreagent is available. Examples of suitable epitopes include the myc andV-5 reporter genes; expression vectors useful for recombinant productionof fusion polypeptides having these epitopes are commercially available(e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-His andpcDNA3.1/V5-His are suitable for expression in mammalian cells).

Additional expression vectors suitable for attaching a tag to the fusionproteins of the invention, and corresponding detection systems are knownto those of skill in the art, and several are commercially available(e.g., FLAG” (Kodak, Rochester N.Y.). Mother example of a suitable tagis a polyhistidine sequence, which is capable of binding to metalchelate affinity ligands. Typically, six adjacent histidines are used,although one can use more or less than six. Suitable metal chelateaffinity ligands that can serve as the binding moiety for apolyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli. E.(1990) “Purification of recombinant proteins with metal chelatingadsorbents” In Genetic Engineering: Principles and Methods, J. K.Setlow, Ed., Plenum Press, NY; commercially available from Qiagen (SantaClarita, Calif.)).

Various aspects of the invention are further described and illustratedby way of the several examples which follow, none of which are intendedto limit the scope of the invention.

EXAMPLES Example 1 Generation and Characterization of Evolved ConsensusGreen Fluorescent Proteins Materials and Methods:

CDR3 Insertions into CGP

The 60-bp CDR3 sequences were inserted into CGP [SEQ ID NO: 27] by PCRassembly. The PCR primers generally contained a 20-bp long CGP specificsequence and a 40-bp 5′ tail encoding one part of the CDR3. The two CDR3containing primers had a 20-bp homologous overlapping sequence tofacilitate PCR assembly. The following general procedure was used,unless otherwise described. The reaction was performed in 50 μlcontaining 1× Thermopol buffer (NEB), 250 μM dNTP, 0.5 μM of eachprimers and 1.25 U Taq polymerase (NEB) with cycling conditions asfollows: 1 min initial denature at 94° C., then 30 cycles of 94° C. 15sec, 55° C., 15 sec, 30 sec, 72° C., then a final elongation of 5 min at72° C. TABLE IV contains the primer sequences. The nucleotide sequenceencoding the 20 amino acid long CDR3 sequence was different for eachinsert, using different codons, in order to prevent homologousrecombination in the bacteria. Inserting single CDR3s into CGP [SEQ IDNO: 27] was achieved by performing two PCR reactions with 1) CGP-5′ andCDR-loop-x-R-CGP and 2) CDR-loop-x-F-CGP and CGP-3′ primers. The xdenotes the loop number. The resulting bands were gel purified andassembled in an assembly reaction with CGP-5′ and CGP-3′ primers addedafter 25 cycles.

Multiple CDR3s were inserted similarly. The double inserted librarieswere assembled from 3 fragments; the triple inserted libraries used 4fragments. For example the double library containing CDR3 insert in loop1 and loop 3 were assembled from fragments amplified using theseprimers; 1) CGP-5′ and CDR-loop-1-R-CGP; 2) CDR-loop1-F-CGP andCDR-loop-3-R-CGP; 3) CDR-loop-3-F CGP and CGP-3′ (see TABLE IV). Otherlibraries were created similarly using the appropriate primers.

DNA Shuffling

DNA shuffling was performed according to Zhao, 1997⁹¹. Briefly, 10 μg oftemplate DNA, CGP [SEQ ID NO: 27] containing 1, 2 or 3 CDR3 inserts,were digested with 1 U of Dnasel (NEB) for 10 minutes at 15° C. in 50 mMTris-Ac pH 7.5, 2 mM CoCl₂. The reaction was terminated by heating for 3minutes at 90° C., and DNA fragments purified by spin-columnchromatography on Sephadex-25 (GE Healthcare) columns. The digestedtemplate was assembled in a primerless PCR reaction with 1.25 U Pfu Exo⁻DNA polymerase (Stratagene) using 15 μl of the digested template in abuffer containing 1×Pfu reaction buffer, 0.4 mM dNTP in a 25 μl reactionvolume. The cycling conditions were 97° C., 3 min initial denature, then35 cycles of 96° C. 25 sec, 56° C. 25 sec, 72° C. 1 min, with a finalextension for 5 min at 72° C. 3 μl of the amplification reaction wasamplified by 2.5 U Tag polymerase (NEB) in a 100 μl reaction containing1× Thermopol buffer (NEB), 250 μM dNTP, 0.5 μM of CGP-5′ and CGP-3′primers with the following cycling; 1 minute initial denature at 94° C.,then 30 cycles of 94° C., 15 see; 60° C., 15 sec; 72° C. 30 sec; with afinal elongation for 5 minutes at 72° C. The PCR product wasphenol/chloroform extracted and purified by spin-columns containingSephadex G-75 (GE Healthcare). The purified DNA was digested with BssHII(NEB) and NheI (NEB) according to the manufacturers recommendation andcloned into pETCK3 (Kiss et al., 2006⁸⁸). The ligation waselectroporated into BL21(DE3) Gold electrocompetent cells. The cellswere plated on nitrocellulose filters on LB agar plates containing 50μg/ml kanamycin and 3% glucose and grown overnight at 37° C. The filterswere transferred onto kanamycin LB plates containing 1 μg/ml IPTG andinduced for 4 hours at 30° C. Colonies that were greenest afterinduction were picked and sequenced. The selected clones for the nextround of shuffling were pooled and the CDR3 sequences were recreated byPCR assembly using CDR3 specific primers that lacked any CGP [SEQ ID NO:27] specific sequences.

Protein Expression and Purification

Plasmids encoding the fluorescent proteins cloned into pETCK3 weretransformed into E. coli BL21 DE3 cells (Stratagene). Single colonytransformants were cultured overnight at 37° in Luria Broth with 50μg/ml kanamycin. The overnight cultures were suspended in fresh TerrificBroth containing 50 μg/ml Kanamycin and transferred to the KalypsisAirlift Fermentation. System, based on the system described by Lesley etal⁸⁹. Cultures were grown at 37° for 3 hours (optical density of 1.5-2.5(600 nm)) on 100% air. The temperature was reduced to 30° and IPTG addedto a final concentration of 1 mM. After 4 hour of growth, 50% air and50% oxygen, cells were harvested by centrifugation and the resultingpellets were stored overnight at −20°. The bacteria pellets were removedfrom storage, thawed, and suspended in lysis buffer (500 mM NaCl, 5 mMImidazole). Cells were lysed by sonication in the Kalypsis pre-chilledrotor, using 4 cycles of one minute sonication (duty cycle 100,amplitude 75) followed by one minute rest, then centrifuged at 7000×gfor 30 min. The Kalypsis Robot transferred the supernatant to the nickelcolumns (Nickel. Chloride bound to GE Chelating Sepharose Fast FlowResin) which were washed with (500 mM NaCl, 5 mM Imidazole). The boundproteins were eluted with (500 mM NaCl, 500 mM Imidazole).

The fluorescence of the purified proteins was measured (SPECTRAFluorPlus, 492 nm, optimal gain 44) in arbitrary fluorescence units measuredat 535 nm. An SDS-PAGE gel was loaded with samples based on equalfluorescence and proteins were quantified against protein standards,using the Syngene GeneTool Software.

Thermostability Measurements

Proteins of equal fluorescence were diluted into 50 μl of TNG buffer(100 mM Tris-Ac pH 7.5, 100 mM NaCl, 10% glycerol) and placed into 0.2ml thin wall PCR tubes. Thermal cyclings were performed in a Rotor-Gene6000 real time PCR machine (Corbett Life Science). Fluorescence and gainwere adjusted so that the fluorescence of the starting samples wasbetween 90-100. The melting profile was resolved between 30° C. and 99°C. Temperature was raised by 0.5° C. increments. The samples wereincubated at each temperature for 60 sec.

Single Molecule Spectroscopy

Fluorescence Correlation Spectroscopy was performed in the same setupdescribed previously⁴⁴. Quantum yield was determined relative toFluorescein from the ratio of integrated fluorescence signal to theabsorbance at 488 nm.

Chemical Denaturation

Equilibrium fluorescence values were measured by diluting guanidinehydrochloride denatured eCGP variants into TNG containing 5 mM DTT tovarious final guanidine concentrations between 1 and 8 M in incrementsof 0.15 M guanidine, and allowing refolding to proceed at 15° C.Fluorescence values were measured using a FL600 Microplate FluorescenceReader (488-nm excitation, 530-nm emission, 10-nm band pass) and scaledby dividing by the fluorescence levels of corresponding nondenaturedsamples diluted in parallel as a reference. Midpoint recoveryconcentrations of guanidine Cm (recovery of 50% of the initialfluorescence) were determined from sigmoidal fits using SOLVER in EXCEL,to the scaled fluorescence value F using the equation Fj 1/4a+b/(1+(Cj/Cm)h), where a, b, Cm and h are adjustable parameters, and Cjis the molarity of the guanidine in the refolding experiment j. The datawere used to calculate the dependence of the standard free energy ofdenaturation, DG1 1/4−RT In K, on guanidine concentration, where R isthe gas constant, T is the absolute temperature and K is the equilibriumconstant, which can be calculated from the experimental data by usingthe standard equation K 1/4 [(y)N−(y)]/[(y)−(y)D], where (y) is theobserved value of the parameter used to follow unfolding, and (y)N and(y)D are the (y) values for the native state and the denatured state,respectively, under the same conditions under which (y) was measured.

Results Evolutionary Strategy

A recursive evolutionary strategy was employed, in which single insertswere grafted into exposed loops in such a way that upon each insertion,folding and function were significantly affected but not destroyed (FIG.1A). This provides a baseline which may be improved by evolution. Uponovercoming the effect of a single insert, the procedure is then repeatedwith a second, and finally a third insert. In this way it is possible toovercome a final destabilizing force that would completely destroy bothfolding and function if applied in a single step.

This method was applied to CGP (SEQ ID NO:27] by modeling the structureof this protein on that of Dronpa, the closest fluorescent protein forwhich a structure has been determined (Wilmann et al., 2006⁹⁰), andtargeting three identified beta turns for insertion. These were termedloop 1 (V18/N19), loop 2 (E96/097) and loop 3 (E164/G165). Thedestabilizing insert used was based on a human heavy chain thirdantibody omplementarity determining region (HCDR3) sequence. AlthoughHCDR3s are highly diverse loops, they are embedded into a relativelyconserved beta sheet structure, as a result of which the amino acids ateither end (cysteine 104 and tryptophan 119; IMGT numbering⁶⁷ are alwaysjoined by two hydrogen bonds. As the usual distance between these twoamino acids is similar to that between amino acids just before the turnsdescribed above, it was presumed that the insertion of such a sequenceinto a CGP loop would probably be disruptive to folding, but would notdestroy it completely. In order to avoid the presence of an unpairedcysteine (the HCDR3N terminal cysteine normally disulfide bonds withanother cysteine in framework one), this codon was mutated to a serine,which is able to form the same hydrogen bonds. The final sequence used(SARSFYLQSDLAAGDFDSWG) [SEQ ID NO: 26] based on a randomly picked HCDR3with a few internal changes to facilitate cloning, was inserted atV18/N19 and E96/D97 in two independent PCR assemblies. As expected, thisresulted in a significant reduction in the fluorescence of inducedbacterial colonies as shown in FIG. 1B.

After three rounds of error prone PCR, and DNA shuffling on these twomodified genes, the fluorescence of induced bacterial colonies increasedsignificantly, some reaching the levels of the original CGP protein(FIG. 1B). During the mutation and selection process, the gene wasalways reassembled using the HCDR3 as an anchor, in order to forcemutations into the CGP and not the inserted HCDR3. After three rounds,PCR assembly was again used to insert the same HCDR3 amino acid sequenceat position E164/G165 of the genes from 23 fluorescent evolved clonescontaining an insert at V18/N19 and 22 clones containing an insert atE96/D97. The DNA sequence encoding the HCDR3 insert was altered to avoidrecombination with the first insert. A significant reduction influorescence was again observed, which could be restored after threefurther rounds of evolution, carried out as before by PCR assembly ofnow three fragments using the two HCDR3 inserts as anchors.

The process was repeated a final time, pooling fluorescent colonies andinserting the HCDR3 into three sites (V18/N19, E96/D97 and E164/G165).With the proteins containing three inserts, four rounds of evolutionwere required before fluorescence was significantly restored. After eachround of evolution approximately 100 clones were sequenced, allowinganalysis of the accumulated mutations (FIG. 2). It should be pointed outthat although the use of assembly PCR to insert each additional loopinto CGP [SEQ ID NO: 27] allowed mutations accumulated in previousrounds to persist into the following evolutionary rounds, this was nottrue of those mutations close to the insert site, which were“overridden” by the primers used for insertion in the first round. Insubsequent rounds, the HCDR3 insertion sequences themselves were usedfor assembly, allowing reappearance of mutations close to insertions. Ingeneral 4 classes of mutations were observed: 1) those (e.g. D7E, M40L,T59P, V60A) appearing immediately and retained throughout; 2) thosewhich first appear with a single insert, are specific for that insert(e.g. 098H for 18/19 inserts, and K22E for 96/97 inserts), are retainedin the presence of two inserts, but are then lost when three inserts arepresent; 3) those appearing in the presence of two inserts, andpersisting in the presence of three inserts (e.g. E164K, K190E, K208R);and 4) those (e.g. A175, K301, F34Y, A53S) which are only found whenthree inserts are present.

Gene Synthesis

Genes corresponding to the proteins without inserts were synthesized(Blue Heron Biotechnology) for each of the five evolutionary paths (FIG.1A). Synthesized genes contained those mutations that led to amino acidchanges in at least 20% of sequences, and silent mutations found ingreater than 90% of sequences. In addition, one silent mutationfrequently found adjacent to a non-silent mutation was also included. Inorder to concentrate on mutations responsible for global increases instability, rather than mutations responding to specific changes insecondary structure adjacent to the insert site, those mutations foundwithin two amino acids of an insertion point were not included, eventhough there are examples of mutations in loops (e.g. Y39N in sfGFP⁷)which are globally

The aligned amino acid sequences of the final genes synthesized,compared to CGP [SEQ ID NO: 27], are shown in FIG. 3. As can be seen,some mutations (eight of eighteen) recapitulate amino acids found influorescent proteins used to create the CGP consensus sequence. Theremaining ten mutations are equally split between those found in mAG(and modified for CGP) and those not previously found in any otherfluorescent protein, and unique to these evolved proteins. Of themutations which revert back to mAG, three (D7E, M40L, A69T) are found inmost of the evolved proteins, while the remaining two (K32N and F34Y)are each found in only one or two of the proteins. The reversion of suchpresumably destabilizing mutations in consensus sequences is similar tothose found in other examples³⁷⁻⁴⁰, and underlie the importance ofexamining the roles of individual amino acids for their contributions tostability.

Properties of eCGPs

The five fluorescent protein genes were cloned into pETCK3⁸⁸ andexpressed in BL21. All were able to direct the synthesis of fluorescentproteins at levels comparable to, or exceeding, CGP and mAG (FIG. 4 a).The excitation/emission properties (FIG. 4 b and TABLE I) of theproteins were similar to either CGP [SEQ ID NO: 27] (eCGP1 [SEQ ID NO:6] and eCGP2 [SEQ ID NO: 7]) or mAG (eCGP13 [SEQ ID NO: 8], eCGP23 [SEQID NO: 9] and eCGP123 [SEQ ID NO: 10), with the CGP series beingslightly red shifted compared to the mAG series. The quantum yields ofthe proteins ranged from 0.54 (eCGP1) [SEQ ID NO: 6] to 0.75 (eCGP13)[SEQ ID NO: 8], not too dissimilar to that of mAG (0.83). All proteinswere monomeric as determined by gel filtration (not shown) orfluorescence correlation spectroscopy (TABLE I).

In a first test of protein thermostability, the proteins were slowlymelted at 0.5° C./min, using a real time PCR machine (Rotor-Gene 6000,Corbett Life Sciences, FIG. 5 a) which monitored fluorescence changeswith temperature in real time. The temperature was gradually increasedto 99° C., and then returned to 30° C., to monitor recovery. Afterapproximately 38° C., all proteins showed a reduction in fluorescentwith increasing temperature as shown in FIG. 5 a. This fluorescence lossis characteristic of fluorescent proteins, and thought to be due to twocomponents: changes in the immediate fluorophore environment caused byincreased thermal vibrations, and unfolding of the proteins.Fluorescence loss due to the former are immediately reversible and donot represent unfolding^(68, 69), while fluorescence loss due to thelatter require refolding for fluorescence to return. As temperatureincreases, the proportion of fluorescence loss due to these twocomponents will vary, depending upon the stability of the protein andthe temperature. In general, little of the fluorescence loss is causedby unfolding until the temperature at which cooperative unfolding startsis reached. This is recognized as an inflection point in the meltingcurve, and represents the point at which unfolding suddenly accelerates.This is similar to changes in CD spectra observed with increasingtemperature).

All proteins, with the exception of eCGP23 [SEQ ID NO: 9] and eCGP123[SEQ ID. NO: 10], showed cooperative unfolding as the temperature wasincreased, with inflection points between 73 and 87° C., cooperativetransition midpoints two to three degrees later, and characteristicsteeper denaturation curves⁶⁹, eCGP23 [SEQ ID NO: 9] and eCGP123 [SEQ IDNO: 10] were characterized by the absence of a clear cooperativetransition, and even at 99° C., some fluorescence remained (FIG. 5 b).Recovery upon cooling to 30° C. resulted in essentially complete (96%)recovery of eCGP123 [SEQ ID NO: 10], and 85% recovery of eCGP23 [SEQ IDNO: 9] (TABLE II). The remaining proteins recovered to varying degrees,depending upon the degree of evolution. For all the evolved proteins,54-61% of the fluorescence recovery occurred instantaneously, while formAG and CGP, the instant recovery was lower (35% and 44% respectively).

The same order of stability was observed when the proteins were treatedwith multiple heat cool cycles (equivalent to 60 “PCR cycles” with 1minute denaturation at 99° C. and 2 minutes recovery at 30° C. —FIG. 5 cfor CGP [SEQ ID NO: 27] and eCGP123 [SEQ ID NO: 10]). eCGP123 [SEQ IDNO: 10] and eCGP23 [SEQ ID NO: 9] continued to show low levels offluorescence at 99° C., while the other proteins rapidly lostfluorescence at this temperature. After 60 heat/cool cycles, and at eachreturn to 30° C., the fluorescence of the two stable proteins returnedto their pretreatment levels, while the remaining proteins showed adramatic drop after the first heat cycle, with fluorescence furtherdecreasing to zero with additional cycles, and little recovery uponreturn to 30° C.

One last test of thermal stability was the ability of the proteins toresist high temperature for prolonged periods. The proteins were allheated to 80° C. or 85° C. This resulted in the initial rapid loss ofover 80% fluorescence due to thermal vibration, which stabilized afterabout six to seven minutes. The fluorescence of the different proteinswas normalized at this time (arrow FIG. 5 d), and further fluorescenceloss monitored for 14 hours. eCGP123 [SEQ ID NO: 10] and eCGP23 [SEQ IDNO: 9] lost approximately 15% fluorescence after 14 hours at 80° C.,while all the other proteins, with the exception of eCGP13 [SEQ ID NO:8] which was intermediate, had lost all fluorescence by 2-3 hours (FIG.5 e). At 85° C. the fluorescence loss of the less stable proteins. (CGP[SEQ ID NO: 27[, eCGP1 [SEQ ID NO: 6] and mAG) was complete by fiveminutes. eCGP1 [SEQ ID NO: 6] and eCGP13 [SEQ ID NO: 8] showed completeloss of fluorescence by three hours, while after 14 hours eCGP23 [SEQ IDNO: 9] and eCGP123 [SEQ ID NO: 10] still retained approximately 10-15%of the normalized fluorescence at 85° C. (FIG. 5F).

Thermal denaturation was monitored using measures independent ofintrinsic fluorescence. However, the Thermofluor assay^(71, 72) wasunsuccessful due to degradation of the Sypro Orange at temperaturesabove 80° C., and it also proved impossible to carry out circulardichroism at the high temperatures required.

eCGP stability was also studied by denaturation in guanidinehydrochloride (FIG. 6A and TABLE III) with unfolding monitored byfluorescence. At equilibrium, which required over two weeks, eCGP123[SEQ ID NO: 10] and eCGP23 [SEQ ID NO: 9] were again the most stableproteins, with melting (kd) occurring at 6.45 M guanidine for eCGP123[SEQ ID NO: 10] and 6.19 M for eCGP23 [SEQ ID NO: 9]. However, the orderof stability for the remaining proteins was slightly different to thatobserved with thermal denaturation, with eCGP2 [SEQ ID NO: 7] beingsignificantly more stable than eCGP13 [SEQ ID NO: 8], and CGP [SEQ IDNO: 27] being more stable than eCGP1 [SEQ ID NO: 6]. By extrapolating anatural log fit of the sigmoidal denaturation curve to infinite dilution(FIG. 6B), the ΔG was determined, which again showed eCGP123 [SEQ ID NO:10] to be by far the most stable protein at 12.4 kcal/mol.

CGP [SEQ ID NO: 27], mAG, and eCGP123 [SEQ ID NO: 10], representing thestarting, evolved, and closest natural proteins, were also analyzed forfolding kinetics. Proteins were denatured in Gdn HCl, and fluorescencerecovery monitored upon dilution into fresh buffer. Although CGP [SEQ IDNO: 27] is much less stable than mAG, it displayed an approximately3.5-fold faster initial rate for fluorescence recovery relative to themore stable mAG (FIG. 6C, inset). This faster folding behavior isconsistent with the observation that CGP also unfolds much faster thanmAG in 8 M Gdn HCl as noted above. Such behavior is typical of simpletwo-state folders, for which increased forward folding rate is mirroredby a corresponding increased unfolding rate.

eCGP123 [SEQ ID NO: 10] folds 4-fold faster than CGP [SEQ ID NO: 27].The increased stability of eCGP123 [SEQ ID NO: 10] relative to CGP [SEQID NO: 27] likely results from the very slow unfolding of eCGP123 [SEQID NO: 10] consistent with the slow approach to equilibrium during theequilibrium Gdn HCl unfolding experiments and the thermal stability.

Tables

TABLE I Absorption Emission max (nm) max (nm) Q.Y. R₀, nm CGP [SEQ ID503 515 0.66 20.3 NO: 27] eCGP1 [SEQ ID 504 514 0.54 19.3 NO: 6] eCGP2[SEQ ID 501 511 0.59 22.9 NO: 7] eCGP13 [SEQ ID 493 505 0.75 20.9 NO: 8]eCGP23 [SEQ ID 493 504 0.73 16.6 NO: 9] eCGP123 [SEQ ID 493 504 0.6921.3 NO: 10] mAG 491 505 0.83 18.6

TABLE II RECOVERY AFTER THERMAL MELT instant recovery % instant % finalas % of total recovery recovery recovery eCGP123 [SEQ ID 58.8 96.0 61.3NO: 10] eCGP23 [SEQ ID 47.7 85.5 55.8 NO: 9] eCGP13 [SEQ ID 33.4 55.260.5 NO: 8] eCGP2 [SEQ ID 12.0 22.3 53.8 NO: 7] eCGP1 [SEQ ID 15.0 27.055.6 NO: 6] CGP [SEQ ID 8.0 18 44.4 NO: 27] mAG 7.5 21.3 35.2

TABLE III STABILITY BY GUANIDINE DENATURATION kd [GnHCl] h ΔG(H₂O) m CGP[SEQ ID 2.56 9.71 4.9 ± 0.1 1.9 ± 0.05 NO: 27] eCGP1 [SEQ ID 2.12 12.456.1 ± 0.0 2.9 ± 0.02 NO: 6] eCGP2 [SEQ ID 5.12 15.53 8.5 ± 0.5 1.7 ±0.10 NO: 7] eCGP13 [SEQ ID 3.15 11.08 5.9 ± 0.1 1.9 ± 0.04 NO: 8] eCGP23[SEQ ID 6.19 14.43 9.8 ± 1.4 1.6 ± 0.23 NO: 9] eCGP123 [SEQ ID 6.4514.29 12.4 ± 2.2  2.0 ± 0.33 NO: 10] mAG 5.86 16.66 8.9 ± 0.9 1.5 ± 0.15

TABLE IV OLIGONUCLEOTIDES USED Name Oligo Sequence CDR3-loop1-FCTTGCAATCCGATCTTGCAGCAGGTGACTTCGACTCTTGGGGT [SEQ ID NO: 11]CDR3-loop1-F-CTTGCAATCCGATCTTGCAGCAGGTGACTTCGACTCTTGGGGTAACGGCCATAAATTTGTAATTG CGP[SEQ ID NO: 12] CDR3-loop1-R CACCTGCTGCAAGATCGGATTGCAAGTAGAAGCTACGAGCACT[SEQ ID NO: 13] CDR3-loop1-R-CACCTGCTGCAAGATCGGATTGCAAGTAGAAGCTACGAGCACTAACGGCACCTTCCATACGC CGP[SEQ ID NO: 14] CDR3-loop2-F CCTCCAAAGTGACTTAGCTGCCGGCGATTTTGATAGCTGGGGC[SEQ ID NO: 15] CDR3-loop2-F-CCTCCAAAGTGACTTAGCTGCCGGCGATTTTGATAGCTGGGGCGATCAAGGAATTTGTATCGC CGP[SEQ ID NO: 16] CDR3-loop2-R CGCCGGCAGCTAAGTCACTTTGGAGGTAAAATGAGCGGGCCGA[SEQ ID NO: 17] CDR3-loop2-R-CGCCGGCAGCTAAGTCACTTTGGAGGTAAAATGAGCGGGCCGATTCATAGGTCATAGAGCGTTC CGP[SEQ ID NO: 18] CDR3-loop3-F TTTACAGTCTGACTTGGCGGCTGGGGATTTCGATTCGTGGGGG[SEQ ID NO: 19] CDR3-loop3-F-TTTACAGTCTGACTTGGCGGCTGGGGATTTCGATTCGTGGGGGGGAGGTGGACACTACCGCTG CGP[SEQ ID NO: 20] CDR3-loop3-R CCCCAGCCGCCAAGTCAGACTGTAAATAGAAAGACCGCGCAGA[SEQ ID NO: 21] CDR3-loop3-R-CCCCAGCCGCCAAGTCAGACTGTAAATAGAAAGACCGCGCAGATTCGAGCAGAAGTGCCATG CGP[SEQ ID NO: 22] CGP-3′ TTTGCCGCTAGCTTTAGCCTGAGACGGTAACATAGAATAGC[SEQ ID NO: 23] CGP-5′ TACATATGGGCGCGCATGCCTCAGTAATTAAACCG[SEQ ID NO: 24]

TABLE OF DNA AND PROTEIN SEQUENCES eCGP DNA Sequences: eCGP1[SEQ ID NO: 1] 1   ATGTCAG TAATTAAACC GGAAATGAAA ATTAAATTGC GTATGGAAGG TGCCGTTAACGGCCATAAAT TTGTAATTGA AGGGGAAGGA AAAGGCAACC 98CATTCGAAGG AACCCAGACC CTGGATTTAA CCGTAAAAGA AGGCGCACCT CTCCCTTTCCCGTACGACAT CCTCACCCCA CTCTTCCAAT ACGGCAATCG 198CGCTTTCGCC AAATACCCAC AAGATATTCC AGACTATTTT AAACAAACAT TCCCCGAAGGCTATTCTTGG GAACGCTCTA TGACCTATGA AGATCATGGA 298ATTTGTATCG CTACCTCCGA CATTACTATG GAAGGCGACT GTTTTATTTA TAAAATTCGCTTTGATGGAA CTAACTTCCC CCCGAACGGC CCTGTAATGC 390AAAAGAAGAC CTTAAAATGG GAACCTAGCA CCGAAAAAAT GTATGTACGC GACGGAGTTCTTAAGGGTGA CGTAAACATG GCACTTCTGC TCGAAGGAGG 498TGGACACTAC CGCTGCGATT TTAAAACCAC TTATAAAGCC AAAAAAGATG TTCGTCTTCCAGATGCACAC AAGGTGGACC ACCGCATTGA AATCCTGAGC 598CACGATAAAG ATTATAATAA AGTTAAACTC TATGAACACG CCGAAGCCCG CTATTCTATGTTACCGTCTC AGGCTAAAGC TAGC eCGP2 [SEQ ID NO: 2] 1   ATGTCAG TAATTAAACC GGAAATGAAA ATTAAATTGC GTTTGGAAGG TGCCGTTAACGGCCATGAAT TTGTAATTGA AGGAGAAGGA AAAGGCAAAC 98CATTCGAAGG AACCCAGACC CTGGATTTAA CCGTAAAAGA AGGCGCACCT CTCCCTTTCGCGTACGACAT CCTCACCCCA GCCTTCCAAT ACGGCAATCG 198CGCTTTCGCC AAATACCCAA AAGATATTCC AGACTATTTT AAACAAACAT TCCCCGAAGGCTATTCTTGG GAACGCTCTA TGACCTATGA AGATCAAGGA 298ATTTGTATCG CTACCTCCGA CATTACTATG GAAGGAGACT GTTTTTTTTA TAAAATTCGCTTTGATGGAA CTAACTTCCC CCCGAACGGC CCTGTAATGC 398AAAAGAAGAC CTTAAAATGG GAACCTAGCA CCGAAAAAAT GTATGTACGC GACGGAGTTCTTAAGGGTGA CGTAAACATG GCACTTCTGC TCGAAGGAGG 498TGGACACTAC CGCTGCGATT TTAAAACCAC TTATAAAGCC AAAAAAGATG TTCGTCTTCCAGATGCACAC AAGGTGGACC ACCGCATTGA AATCCTGAGC 598CACGATAAAG ATTATAATAA AGTTAAACTC TATGAACACG CCGAAGCCCG CTATTCTATGTTACCGTCTC AGGCTAAAGC TAGC eCGP13 [SEQ ID NO: 3] 1   ATGTCAG TAATTAAACC GGAAATGAAA ATTAAATTGC GTATGGAAGG TGCCGTTAACGGCCATAAAT TTGTAATTGA AGGGGAAGGA AAAGGCAACC 98CATTCGAAGG AACCCAGACC CTGGATTTAA CCGTAAAGGA AGGCGCACCT CTCCCTTTCGCGTACGACAT CCTCACCCCA GTCTTCCAAT ACGGCAATCG 198CGCTTTCACC AAATACCCAC AAGATATTCC AGACTATTTT AAACAAACAT TCCCCGAAGGCTATTCTTGG GAACGCTCTA TGACCTATGA AGATCATGGA 298ATTTGTATCG CTACCTCCGA CATTACTATG GAAGGCGACT GTTTTATTTA TAAAATTCGCTTTGATGGAA CTAACTTCCC CCCAAACGGC CCTGTAATGC 398AAAAGAAGAC CTTAAAATGG GAACCTAGCA CCGAAAAAAT GTATGTACGC GACGGAGTTCTTAAGGGTGA CGTAAACATG GCACTTCTGC TCGAAGGAGG 498TGGACACTAC CGCTGCGATT TTAAAACCAC TTATAAAGCC AAAAAAGATG TTCGTCTTCCAGGTGCACAC AAGGTGGACC ACCGCATTGA AATCCTGAGC 598CACGATAAAG ATTATAATAA AGTTAAACTC TATGAACACG CCGAAGCCCG CTATTCTATGTTACCGTCTC AGGCTAAAGC TAGC eCGP23 [SEQ ID NO: 4] 1   ATGTCAG TAATTAAACC GGAAATGAAA ATTAAATTGC GTTTGGAAGG TGCCGTTAACGGCCATGAAT TTGTAATTGA AGGAGAAGGA AAAGGCAAAC 98CATTCGAAGG AACCCAGACC CTGGATTTAA CCGTAAAAGA AGGCGCACCT CTCCCTTTCGCGTACGACAT CCTCACCCCA GCCTTCCAAT ACGGCAATCG 198CGCTTTCACC AAATACCCAA AAGATATTCC AGACTATTTT AAACAAACAT TCCCCGAAGGCTATTCTTGG GAACGCTCTA TGACCTATGA AGATCAAGGA 298ATTTGTATCG CTACCTCCGA CATTACTATG GAAGGAGACT GTTTTTTTTA TAAGATTCGCTTTGATGGAA CTAACTTCCC CCCGAACGGC CCTGTAATGC 398AAAAGAAGAC CTTAAAATGG GAACCTAGCA CCGAAAAAAT GTATGTACGC GACGGAGTTCTTAAGGGTGA CGTAAACATG GCACTACTGC TCGAAGGAGG 498TGGACACTAC CGCTGCGATT TTAAAACCAC TTATAAAGCC AAAAAAGATG TTCGTCTTCCAGATGCACAC GAGGTGGACC ACCGCATTGA AATCCTGAGC 598CACGATAAAG ATTATAATAA AGTTAAACTC TATGAACACG CCGAAGCCCG CTATTCTATGTTACCGTCTC AGGCTAAAGC TAGC eCGP123 [SEQ ID NO: 5] 1   ATGTCAG TAATTAAACC GGAAATGAAA ATTAAATTGC GTATGGAAGG TGCCGTTAACGGCCATAAAT TTGTAATTGA AGGAGAAGGA ATAGGCAAAC 98CATACGAAGG AACCCAGACC CTGGATTTAA CCGTAAAAGA AGGCGCACCT CTCCCTTTCTCGTACGACAT CCTCACCCCA GCCTTCCAAT ACGGCAATCG 198CGCTTTCACC AAATACCCAA AAGATATTCC AGACTATTTT AAACAAGCAT TCCCCGAAGGCTATTCTTGG GAACGCTCTA TGACCTATGA AGATCAAGGA 298ATTTGTATCG CTACCTCCGA CATTACTATG GAAGGAGACT GTTTTTTTTA TAAGATTCGCTTTGATGGAA CTAACTTCCC CCCGAACGGC CCTGTAATGC 398AAAAGAAGAC CTTAAAATGG GAACCTAGCA CCGAAAAAAT GTATGTACGC GACGGAGTTCTTAAGGGTGA CGTAAACATG GCACTTCTGC TCGAAGGAGG 498TGGACACTAC CGCTGCGATT TTAAAACCAC TTATAAAGCC AAAAAAGATG TTCGTCTTCCAGATGCACAC GAGGTGGACC ACCGCATTGA AATCCTGAGC 598CACGATAAAG ATTATAATAA AGTTAGACTC TATGAACACG CCGAAGCCCG CTATTCTATGTTACCGTCTC AGGCTAAAGC TAGC eCGP Amino Acid Sequences: eCGP1[SEQ ID NO: 6]MSVIKPEMKIKLRMEGAVNGHKFVIEGEGKGNPFEGTQTLDLTVKEGAPLPFAYDILTPVFQYGNRAFAKYPQDIPDYFKQTFPEGYSWERSMTYEDHGICIATSDITMEGDCFIYKIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALLLEGGGHYRCDFKTTYKAKKDVRLPDAHKVDHRIEILSHDKDYNKVKLYEHAEARYSMLPSQAKeCGP2 [SEQ ID NO: 7]MSVIKPEMKIKLRLEGAVNGHEFVIEGEGKGKPFEGTQTLDLTVKEGAPLPFAYDILTPAFQYGNRAFAKYPKDIPDYFKQTFPEGYSWERSMTYEDQGICIATSDITMEGDCFFYKIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALLLEGGGHYRCDFKTTYKAKKDVRLPDAHKVDHRIEILSHDKDYNKVKLYEHAEARYSMLPSQAKeCGP13 [SEQ ID NO: 8]MSVIKPEMKIKLRMEGAVNGHKFVIEGEGKGNPFEGTQTLDLTVKEGAPLPFAYDILTPVFQYGNRAFTKYPQDIPDYFKQTFPEGYSWERSMTYEDHGICIATSDITMEGDCFIYKIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALLLEGGGHYRCDFKTTYKAKKDVRLPGAHKVDHRIEILSHDKDYNKVKLYEHAEARYSMLPSQAKeCGP23 [SEQ ID NO: 9]MSVIKPEMKIKLRLEGAVNGHEFVIEGEGKGKPFEGTQTLDLTVKEGAPLPFAYDILTPAFQYGNRAFTKYPKDIPDYFKQTFPEGYSWERSMTYEDQGICIATSDITMEGDCFFYKIRFDGTNFPPNGPVMQKKTLKWEPSTEKMVVRDGVLKGDVNMALLLEGGGHYRCDFKTTYKAKKDVRLPDAHEVDHRIEILSHDKDYNKVKLYEHAEARYSMLPSQAKeCGP123 [SEQ ID NO: 10]MSVIKPEMKIKLRMEGAVNGHKFVIEGEGIGKPYEGTQTLDLTVKEGAPLPFSYDILTPAFQYGNRAFTKYPKDIPDYFKQAFPEGYSWERSMTYEDQGICIATSDITMEGDCFFYKIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALLLEGGGHYRCDFKTTYKAKKDVRLPDAHEVDHRIEILSHDKDYNKVRLYEHAEARYSMLPSQAK

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1. (canceled)
 2. (canceled)
 3. A nucleic acid molecule comprising apolynucleotide encoding the polypeptide with the amino acid sequence ofSEQ ID NO:
 9. 4. The nucleic acid molecule of claim 3, wherein the aminoacid sequence of SEQ ID NO: 9 is encoded by the polynucleotide of SEQ IDNO:
 4. 5. The nucleic acid molecule of claim 4, which is a vector. 6.The nucleic acid molecule of claim 4, which is an expression vector. 7.(canceled)
 8. (canceled)
 9. A nucleic acid molecule comprising apolynucleotide encoding the polypeptide with the amino acid sequence ofSEQ ID NO:
 10. 10. The nucleic acid molecule of claim 9, wherein theamino acid sequence of SEQ ID NO: 10 is encoded by the polynucleotide ofSEQ ID NO:
 5. 11. The nucleic acid molecule of claim 10, which is avector.
 12. The nucleic acid molecule of claim 10, which is anexpression vector.